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Mass Spectrometry of Macromolecules
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History
• In 1886, Eugen Goldstein observed rays in gas discharges under low
pressure that traveled away from the anode and through channels
in a perforated cathode, opposite to the direction of negatively
charged cathode rays (which travel from cathode to anode).
• Goldstein called these positively charged anode rays
"Kanalstrahlen"; the standard translation of this term into English is
"canal rays".
• Wilhelm Wien found that strong electric or magnetic fields
deflected the canal rays and, in 1899, constructed a device with
parallel electric and magnetic fields that separated the positive rays
according to their charge-to-mass ratio (Q/m).
• Wien found that the charge-to-mass ratio depended on the nature
of the gas in the discharge tube.
• English scientist J.J. Thomson later improved on the work of Wien
by reducing the pressure to create the mass spectrograph.
History
• The first application of mass spectrometry to the analysis of
amino acids and peptides was reported in 1958.
• Carl-Ove Andersson highlighted the main fragment ions
observed in the ionization of methyl esters.
History
• Some of the modern techniques of mass spectrometry
were devised by Arthur Jeffrey Dempster and F.W. Aston in
1918 and 1919 respectively.
• In 1989, half of the Nobel Prize in Physics was awarded to
Hans Dehmelt and Wolfgang Paul for the development of
the ion trap technique in the 1950s and 1960s.
• In 2002, the Nobel Prize in Chemistry was awarded to John
Bennett Fenn for the development of electrospray
ionization (ESI) and Koichi Tanaka for the development of
soft laser desorption (SLD) and their application to the
ionization of biological macromolecules, especially proteins.
In a typical MS procedure
1. A sample is loaded onto the mass spectrometer, and
undergoes vaporization
2. The components of the sample are ionized by one of
a variety of methods (e.g., by impacting them with
an electron beam), which results in the formation of
charged particles (ions)
3. The ions are separated according to their mass-tocharge ratio in an analyzer by electromagnetic fields
4. The ions are detected, usually by a quantitative
method
5. The ion signal is processed into mass spectra
MS instruments consist of three modules
1. An ion source, which can convert gas phase
sample molecules into ions (or, in the case of
electrospray ionization, move ions that exist in
solution into the gas phase)
2. A mass analyzer, which sorts the ions by their
masses by applying electromagnetic fields
3. A detector, which measures the value of an
indicator quantity and thus provides data for
calculating the abundances of each ion present
GENERAL PRINCIPLES: THE PROBLEM
• The ability to determine
accurate molecular masses,
including those of fragments
of large molecules such as
proteins and nucleic acids,
now allows us to identify
molecules from a cellular
mixture, monitor their
changes, and even provide
detailed information on their
structures (from the primary
sequence to potentially the
tertiary fold and quaternary
association of subunits) and
mechanisms of folding.
The three primary components
of a mass spectrometer.
The molecule(s) of interest as a charged ion are placed into the gas phase, then the
molecular species must be separated (or resolved) according to their masses, and, finally,
the molecular ions must be detected.
mass-to-charge (m/Z) ratio
• Mass spectrometry uses an electrostatic potential
to accelerate molecular ions and, thus, the
method does not measure the mass of a
molecule per se, but rather the mass-to-charge
(m/Z) ratio of the molecular ion.
• We will see that molecular ions with smaller
masses or larger numbers of charges will have a
higher velocity, and this can be used as a basis to
resolve various molecular ions according to
differences in their respective masses.
Matrix-assisted laser desportionionization (MALDI)
Ionization of the analyte results from
exchange of electrons and/or protons
(shown here as a proton transition) with
the matrix compound.
In MALDI-TOF-MS, the macromolecule to
be analyzed (the analyte) is first
embedded in a crystal of a matrix
compound, typically a weak organic acid.
The power density required to generate a significant ion
current corresponds to an energy flux of ~20 mJ/cm2.
Matrix for MALDI-TOF
1. 2,5-Dihydroxybenzoic acid (DHB)
 Peptides, proteins, lipids, and oligosaccharides
2. 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid)
 Peptides, proteins, and glycoproteins
3. a-Cyano-4-hydroxycinnamic acid (CHCA)
 Peptides, proteins, lipids, and oligonucleotides
A mass spectrum of myoglobin
Both singly and doubly charged ions are present.
Electrospray ionization (ESI) mass
spectrometry.
RESOLVING MOLECULAR WEIGHTS
BY MASS SPECTROMETRY
Schematic drawing of a time-of-flight
mass spectrometer (TOF-MS)
mass-to-charge (m/Z) ratio
The units of the mass itself may be in atomic mass units (amu) or, for
biological molecules, in daltons (Da, equivalent to 1 gm/mol) or kilodaltons
(kDa equivalent to 1 kg/mol), units that are familiar to biochemists.
Principle of a reflectron in TOF-MS
Magnetic sector mass spectrometry
Only those ions whose angular momentum (as defined
by their mlZ ratios) defines an unimpeded pathway
through the magnetic field will reach the detector.
Quadrupole mass filter
constant electric potential (dc potential)
alternating potential (ac) or a radio frequency (rf) field
The potential at any point (x and y)
within the quadrupole
Any ion coming into the quadrupole will experience a static potential (with
magnitude U) and alternating potential (with a magnitude of V).
Quadrupole mass filter
Notice that the field applies a force of F = 0 along the z-axis
Equations 15.10 and 15.11 can be rewritten in the form of Mathieu‘s differential
equation (which was derived in 1866 to describe the propagation of waves in
membranes) to provide a quantitative prediction of the stability of ions within a
quadrupole field.
Quadrupole filter
low mass
high mass
very heavy ions
The x-z plane acts as a low mass filter while the y-z plane acts
as a high pass filter, leaving only those masses that overlap
between the two to be detected.
DETERMINING MOLECULAR
WEIGHTS OF BIOMOLECULES
ESI-MS spectrum of leucineenkephalin
C13
Na+
ESI-MS of lysozyme
The m/Z ratios of the (M + nH)+n ions are labeled.
ESI-MS
Solving the two simultaneous equations results
in n = 9.0, or that the m/Z = 1590.6 species has
9 protons added.
MW = 14,306.4 Da
IDENTIFICATION OF BIOMOLECULES
BY MOLECULAR WEIGHTS
Strategy for a proteomic experiment using
mass spectrometry and MS fingerprinting
Two-dimensional (2-D) gel electrophoresis
separation of the human prostrate proteome
The proteins identified by mass spectrometric fingerprinting of the tryptic
peptides are labeled.
Protein proteolytic fingerprinting by MS
Trypsin fingerprint of sera transferrin
SEQUENCING BY MASS
SPECTROMETRY
How do we determine the sequence
of a molecule such as a polypeptide
by mass analysis?
Residue Masses (Within a Polypeptide Chain) of
the 20 Common Amino Acids
Residue Masses (Within a Polypeptide Chain) of
the 20 Common Amino Acids
Strategy for assembling a complete polypeptide
sequence from the sequences of proteolytic fragments.
Peptide sequencing by tandem mass
spectrometry (MS/MS).
The fragmentation is accomplished in a collision chamber, which is filled with a neutral gas
(argon or xenon) that breaks the peptide backbone of the peptide-this is called collision-induced
dissociation (CID) and results in a set of product or daughter ions.
Characteristics of the CID Daughter Ions
Daughter ions produced by collisioninduced dispersion (CID)
Proposed mechanism for generation of
B- and Y-ions from CID fragmentation
MS/MS spectrum of the peptide
NFESGK
Nomenclature for CID fragments
CID Mass Spectrum
Note: Not all b or y ions will present in the spectrum
PROBING THREE-DIMENSIONAL
STRUCTURE BY MASS
SPECTROMETRY
Comparison of the hydrogen/deuterium
exchange rates for the protein thioredoxin
Mapping the Core of
hPAP Fibrils by H/D
Exchange
15N-HSQC
No D2O
With D2O
Spectra of hPAP in 90% DMSO
Finding Disorder in Order
When a Crystal Structure Is Not
Enough
Mechanisms for hydrogen/deuterium
exchange in proteins
EXERCISES
EXERCISES